RESUMO
OBJECTIVES: To investigate the effects of autologous serum (AS) and platelet-rich plasma (PRP) on human corneal endothelial cell (HCEC) proliferation and apoptosis in comparison to Y-27632 as the commonly studied Rho-associated kinase (ROCK) inhibitor. METHODS: The human corneal endothelial primary cell line was used for this study. As the treatment groups, HCECs were incubated with AS, PRP, and Y-27632, whereas the control group received no treatment. Cell proliferation (measured by 5-bromo-2'-deoxyuridine [BrdU] incorporation) and apoptosis (based on the caspase-3 level) were compared between the control, Y-27632, AS, and PRP groups. RESULTS: In the Y-27632, AS, and PRP groups, the ratios of BrdU-incorporated cells were significantly higher (115±0.2%, 125±0.2%, 122±0.4% at 24 hr, and 138±2.4%, 160±0.2%, 142±0.2% at 48 hr, respectively) than in the control group (100±18.4% at 24 hr, 100±1.1% at 48 hr) ( P <0.05 for all). Furthermore, AS provided a higher HCEC proliferation ratio compared with the Y-27632 group at 24 and 48 hr ( P <0.05 for all). Caspase-3 was significantly lower in the AS group (60.3±3.3%) than in the control (100±2.3%), Y-27632 (101.9±5.2%), and PRP (101±6.8%) groups ( P <0.05 for all). CONCLUSIONS: The results of this study demonstrated for the first time that AS and PRP promoted HCEC proliferation and AS significantly decreased apoptosis in HCECs. A superior effect on HCEC proliferation was also observed with AS compared with Y-27632. Future "autologous" regenerative therapeutic options for corneal endothelial failure may involve the utilization of AS and PRP owing to their accessibility, simplicity in preparation, immunologic compatibility, and donor-free nature.
Assuntos
Amidas , Plasma Rico em Plaquetas , Piridinas , Humanos , Caspase 3/farmacologia , Bromodesoxiuridina/farmacologia , Células Cultivadas , Proliferação de Células , Regeneração , Células EndoteliaisRESUMO
Adult neurogenesis in the dentate gyrus plays an important role for pattern separation, the process of separating similar inputs and forming distinct neural representations. Estradiol modulates neurogenesis and hippocampus function, but to date no examination of estradiol's effects on pattern separation have been conducted. Here, we examined estrogenic regulation of adult neurogenesis and functional connectivity in the hippocampus after the spatial pattern separation task in female rats. Ovariectomized Sprague-Dawley rats received daily injections of vehicle, 0.32 µg (Low) or 5 µg (High) of estradiol benzoate until the end of experiment. A single bromodeoxyuridine (BrdU) was injected one day after initiation of hormone or vehicle treatment and rats were tested in the delayed nonmatching to position spatial pattern separation task in the 8-arm radial maze for 12 days beginning two weeks after BrdU injection. Rats were perfused 90 min after the final trial and brain sections were immunohistochemically stained for BrdU/neuronal nuclei (NeuN) (new neurons), Ki67 (cell proliferation), and the immediate early gene, zif268 (activation). Results showed that high, but not low, estradiol reduced the density of BrdU/NeuN-ir cells and had significant inter-regional correlations of zif268-ir cell density in the hippocampus following pattern separation. Estradiol treatment did not influence pattern separation performance or strategy use. These results show that higher doses of estradiol can reduce neurogenesis but at the same time increases correlations of activity of neurons within the hippocampus during spatial pattern separation.
Assuntos
Giro Denteado , Hipocampo , Ratos , Feminino , Animais , Ratos Sprague-Dawley , Bromodesoxiuridina/farmacologia , Neurogênese , Estradiol/farmacologiaRESUMO
Newborn neurons from the subventricular zone (SVZ) are essential to functional recovery following ischemic stroke. However, the number of newly generated neurons after stroke is far from enough to support a potent recovery. Adiponectin could increase neurogenesis in the dentate gyrus of hippocampus in neurodegenerative diseases. However, the effect of adiponectin on the neurogenesis from SVZ and the functional recovery after ischemic stroke was unknown, and the underlying mechanism was not specified either. The middle cerebral artery occlusion model of mice was adopted and adiponectin was administrated once a day from day 3 to 7 of reperfusion. The levels of BDNF and p-STAT3 were detected by western blotting on day 7 of reperfusion. The virus-encoded BDNF shRNA with GFAP promoter and a STAT3 inhibitor Stattic were used, respectively. Neurogenesis was evidenced by the expression of doublecortin and 5-bromo-2'-deoxyuridine (BrdU) labelling and brain atrophy was revealed by Nissl staining on day 28 of reperfusion. Neurological functional recovery was assessed by the adhesive removal test and the forepaw grip strength. We found that adiponectin increased both the doublecortin-positive cells and NeuN/BrdU double-positive cells around the injured area on day 28 of reperfusion, along with the improved long-term neurological recovery. Mechanistically, adiponectin increased the protein levels of p-STAT3 and BDNF in astrocytes on day 7 of reperfusion, while silencing BDNF diminished the adiponectin-induced neurogenesis and functional recovery. Moreover, inhibition of STAT3 not only prevented the increase of BDNF but also the improved neurogenesis and functional recovery after stroke. In conclusion, adiponectin enhances neurogenesis and functional recovery after ischemic stroke via STAT3/BDNF pathway in astrocytes.
Assuntos
Isquemia Encefálica , Ataque Isquêmico Transitório , AVC Isquêmico , Acidente Vascular Cerebral , Camundongos , Animais , Ataque Isquêmico Transitório/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Adiponectina/farmacologia , Regulação para Cima , Astrócitos/metabolismo , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Acidente Vascular Cerebral/metabolismo , Neurogênese/fisiologia , Infarto da Artéria Cerebral Média/metabolismo , AVC Isquêmico/metabolismo , Proteínas do Domínio Duplacortina , Isquemia Encefálica/metabolismoRESUMO
In the present study, the in vitro antiproliferative effect of targeting highly expressed cancer protein 1 (Hec1) inhibitor INH1 was investigated in estrogen receptor-positive MCF-7 cell line originating from an in situ carcinoma and triple negative MDA-MB-231 cell line originating from metastatic carcinoma. Cell viability, xCELLigence RTCA DP instrument CI values, MI, BrdU proliferation assay, and AI analyses were employed for this purpose. According to the findings of the current study, INH1 altered cell proliferation by lowering cell viability, CI, MI values, and BrdU proliferation while raising AI values in both cell lines. Between the experimental and control groups, there were noticeable changes (p<0.05). These findings imply that INH1's mode of action is not dependent on the presence of estrogen receptors, making it a potentially effective therapy for breast cancer.
Assuntos
Neoplasias da Mama , Carcinoma , Feminino , Humanos , Apoptose , Neoplasias da Mama/patologia , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Células MCF-7RESUMO
Glioblastoma is one of the deadliest malignant gliomas. Capsaicin is a homovanillic acid derivative that can show anti-cancer effects by regulating various signaling pathways. The aim of this study is to investigate the effects of capsaicin on cell proliferation via ferroptosis in human U87-MG and U251 glioblastoma cells. Firstly, effects of capsaicin treatment on cell viability were determined by MTT analysis. Next, cellular-proliferation and cytotoxicity assays were determined by analyzing bromodeoxyuridine (BrdU) and lactate dehydrogenase (LDH) activity, respectively. Following, acyl-CoA synthetase long chain family member 4 (ACSL4), glutathione peroxidase 4 (GPx4), 5-hydroxyeicosatetraenoic acid (5-HETE), total oxidant status (TOS), malondialdehyde (MDA), total antioxidant status (TAS) and reduced glutathione (GSH) levels were determined by ELISA. Additionally, ACSL4 and GPx4 mRNA and protein levels were analyzed. Capsaicin showed a concentration-dependent anti-proliferative effects in U87-MG and U251 cells. Cell viability was decreased in the both cell lines treated with capsaicin concentrations above 50 µM, while LDH activity increased. Treatment of 121.6, 188.5, and 237.2 µM capsaicin concentrations for 24 h indicated an increase in ACSL4, 5-HETE, TOS and MDA levels in U87-MG and U251 cells (p < 0.05). On the other hand, we found that capsaicin administration caused a decrease in BrdU, GPx4, TAS and GSH levels in U87-MG and U251 cells (p < 0.05). Besides, ACSL4 mRNA and protein levels were increased in the glioblastoma cells treated with capsaicin, whereas GPx4 mRNA and protein levels were decreased. Finally, capsaicin might be used as a potential anticancer agent with ferroptosis-induced anti-proliferative effects in the treatment of human glioblastoma.
Assuntos
Ferroptose , Glioblastoma , Humanos , Glioblastoma/metabolismo , Capsaicina/farmacologia , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Transdução de Sinais , Oxirredução , RNA Mensageiro/metabolismoRESUMO
Despite the decreasing trend in mortality from colorectal cancer, this disease still remains the third most common cause of death from cancer. In the present study, we investigated the antiproliferative and pro-apoptotic effects of (2S,3S,4R)-2-tridecylpyrrolidine-3,4-diol hydrochloride on colon cancer cells (Caco-2 and HCT116). The antiproliferative effect and IC50 values were determined by the MTT and BrdU assays. Flow cytometry, qRT-PCR and Western blot were used to study the cellular and molecular mechanisms involved in the induction of apoptotic pathways. Colon cancer cell migration was monitored by the scratch assay. Concentration-dependent cytotoxic and antiproliferative effects on both cell lines, with IC50 values of 3.2 ± 0.1 µmol/L (MTT) vs. 6.46 ± 2.84 µmol/L (BrdU) for HCT116 and 2.17 ± 1.5 µmol/L (MTT) vs. 1.59 ± 0.72 µmol/L (BrdU), for Caco-2 were observed. The results showed that tridecylpyrrolidine-induced apoptosis was associated with the externalization of phosphatidylserine, reduced mitochondrial membrane potential (MMP) accompanied by the activation of casp-3/7, the cleavage of PARP and casp-8, the overexpression of TNF-α and FasL and the dysregulation of Bcl-2 family proteins. Inhibition of the migration of treated cells across the wound area was detected. Taken together, our data show that the anticancer effects of tridecylpyrrolidine analogues in colon cancer cells are mediated by antiproliferative activity, the induction of both extrinsic and intrinsic apoptotic pathways and the inhibition of cell migration.
Assuntos
Apoptose , Neoplasias do Colo , Humanos , Bromodesoxiuridina/farmacologia , Células CACO-2 , Transdução de Sinais , Neoplasias do Colo/tratamento farmacológico , Proliferação de Células , Linhagem Celular Tumoral , Potencial da Membrana MitocondrialRESUMO
Environmental factors, including pesticide exposure, have been identified as substantial contributors to neurodegeneration and cognitive impairments. Previously, we demonstrated that repeated exposure to deltamethrin induces endoplasmic reticulum (ER) stress, reduces hippocampal neurogenesis, and impairs cognition in adult mice. Here, we investigated the potential relationship between ER stress and hippocampal neurogenesis following exposure to deltamethrin, utilizing both pharmacological and genetic approaches. To investigate whether ER stress is associated with inhibition of neurogenesis, mice were given two intraperitoneal injections of eIf2α inhibitor salubrinal (1 mg/kg) at 24 h and 30 min prior to the oral administration of deltamethrin (3 mg/kg). Salubrinal prevented hippocampal ER stress, as indicated by decreased levels of C/EBP-homologous protein (CHOP) and transcription factor 4 (ATF4) and attenuated deltamethrin-induced reductions in BrdU-, Ki-67-, and DCX-positive cells in the dentate gyrus (DG) of the hippocampus. To further explore the relationship between ER stress and adult neurogenesis, we used caspase-12 knockout (KO) mice. The caspase-12 KO mice exhibited significant protection against deltamethrin-induced reduction of BrdU-, Ki-67-, and DCX-positive cells in the hippocampus. In addition, deltamethrin exposure led to a notable upregulation of CHOP and caspase-12 expression in a significant portion of BrdU- and Ki-67-positive cells in WT mice. Conversely, both salubrinal-treated mice and caspase-12 KO mice exhibited a considerably lower number of CHOP-positive cells in the hippocampus. Together, these findings suggest that exposure to the insecticide deltamethrin triggers ER stress-mediated suppression of adult hippocampal neurogenesis, which may subsequently contribute to learning and memory deficits in mice.
Assuntos
Apoptose , Piretrinas , Camundongos , Animais , Caspase 12/metabolismo , Bromodesoxiuridina/farmacologia , Antígeno Ki-67/metabolismo , Piretrinas/metabolismo , Hipocampo/metabolismo , Neurogênese/fisiologia , Estresse do Retículo EndoplasmáticoRESUMO
Objective: To investigate the effect of photobiomodulation (PBM) on hippocampal neurogenesis, cognitive function, and inflammatory injury in rats with chronic cerebral hypoperfusion. Methods: Bilateral ovariectomy (OVX) was performed on female Sprague-Dawley (SD) rats. One week later, the rats were randomly assigned to three groups, Sham surgery (or Sham) group, bilateral common carotid artery occlusion (BCCAO) group, and PBM intervention (or BCCAO+PBM) group. There were 8 rats in each group. In the BCCAO group, chronic cerebral hyporeperfusion was induced by permanent ligation of bilateral common carotid arteries and no PBM was given. Rats in the Sham group underwent the same surgical procedure except for the occlusion of the two carotids arteries and no PBM was given. In addition to the BCCAO surgery, rats in the BCCAO+PBM group received 808 nm laser therapy (5 min each time at a laser dose of 20 mW/cm 2) of the frontal cortex every other day for 1 month. Between 86 and 90 days after BCCAO, Morris water maze (MWM) was used to observe the spatial learning and memory function of the rats. The rats were sacrificed on day 90 and immunofluorescence staining and Western blot were performed thereafter. Immunofluorescence staining was used to determine the expression of 5-bromodeoxyuracil nucleoside (BrdU), a cell proliferation marker, glial fibrillary acidic protein (GFAP), an astrocyte marker, doublecortin (DCX), a specific marker of newborn neuron precursor cells, NeuN, a marker of mature neurons, and Iba1, a microglia marker, in the hippocampal dentate gyrus (DG) region. Western blot was performed to analyze the protein expressions of inflammasome components, NLRP3, ASC, cleaved caspase-1, and Iba1 in the hippocampus. Results: In the latency trial of MWM test, BCCAO+PBM rats spent shorter periods of time finding the underwater platform than the BCCAO rats did. In the probe trial, after the platform that was original placed in a quadrant was removed, the BCCAO+PBM rats spent longer periods of time exploring the quadrant than the BCCAO animals did ( P<0.05). Compared with BCCAO rats, BCCAO+PBM rats showed significant decrease in the immunofluorescence intensities of GFAP and Iba1 ( P<0.01). PBM intervention significantly increased the number of BrdU-positive cells in the hippocampal DG region compared with those of Sham and BCCAO groups ( P<0.05). Furthermore, the number of NeuN positive cells showed no significant difference among the three groups, while in BCCAO+PBM group, the number of DCX-positive cells was significantly increased ( P<0.001) and the number of DCX +/NeuN + co-located cells was significantly increased compared to that of the BCCAO group ( P<0.001). Compared with those of the BCCAO group, Western blot results showed that the protein expression levels of Iba1, NLRP3, and cleaved caspase-1 in the BCCAO+PBM group were significantly decreased ( P<0.05), while the ASC protein expression level showed no significant difference. Conclusion: PBM can effectively improve the spatial learning and memory function in rats with chronic cerebral hypoperfusion, inhibit the activation of glial cells, reduce inflammatory damage mediated by NLRP3 inflammasome, and promote the regeneration of endogenous neural stem cells in the hippocampal DG region of rats.
Assuntos
Isquemia Encefálica , Inflamassomos , Ratos , Feminino , Animais , Ratos Sprague-Dawley , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Bromodesoxiuridina/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Cognição/fisiologia , Anti-Inflamatórios/farmacologia , Hipocampo , Aprendizagem em Labirinto , Neurogênese , Caspases/metabolismoRESUMO
Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Bromodesoxiuridina/farmacologia , Bromodesoxiuridina/uso terapêutico , Transdução de Sinais , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ágar , Proliferação de Células , Linhagem Celular Tumoral , Apoptose , Histona Desmetilases com o Domínio Jumonji/metabolismoRESUMO
BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.
Assuntos
Neoplasias Ósseas , Neoplasias da Próstata , Receptor PAR-2/metabolismo , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Animais , Neoplasias Ósseas/secundário , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Neoplasias da Próstata/patologia , Receptores Ativados por Proteinase/metabolismoRESUMO
Metoclopramide (MCP) is a drug that has been widely used in recent years due to its hyperprolactinaemia effect on mothers during breastfeeding. The aim of this study was to investigate the proliferative changes that MCP may cause in the maternal breast tissue. In this study, 18 Wistar albino young-adult breastfeeding mothers with their offspring were divided into three groups: control group, low-dose MCP-applied group and high-dose MCP-applied group. The experiment was carried out during the lactation period and at the end of 21 days. Prolactin, BrdU and Ki-67 breast tissue distributions were evaluated by immunohistochemistry, and tissue levels were evaluated biochemically by the ELISA method. According to ELISA and immunohistochemistry results in breast tissue, there was no significant difference between Ki-67 and BrdU results in all groups. Metoclopramide did not change the expression of proliferation molecules Ki-67 and BrdU in breast tissue. These results suggested that while metoclopramide increases breast proliferation, it does not have the risk of transforming the tissue into a tumour.
Assuntos
Lactação , Metoclopramida , Bromodesoxiuridina/farmacologia , Proliferação de Células , Feminino , Humanos , Antígeno Ki-67 , Metoclopramida/efeitos adversosRESUMO
INTRODUCTION: Chronic myeloid leukaemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Drug resistance remains one of the major challenges in CML therapy. MicroRNA (miR)-199a-3p plays an important role in many tumours but has rarely been investigated in CML. We aimed to analyse the role and mechanism of miR-199a-3p in regulating imatinib resistance in CML. METHODS: The expression of miR-199a-3p and mammalian target of rapamycin (mTOR) in the serum of CML patients and CML cells was examined by quantitative real-time polymerase chain reaction. The levels of apoptosis-related proteins were determined using western blot. The relative cell survival rate and cell proliferation were determined using a CCK-8 assay and a bromodeoxyuridine (BrdU) assay, respectively. Cell cycle and apoptosis were analysed using flow cytometry. Moreover, a dual-luciferase reporter assay was performed to verify the correlation between miR-199a-3p and mTOR. RESULTS: MiR-199a-3p was downregulated in the serum of CML patients and in CML cells, while mTOR was upregulated. Both miR-199a-3p overexpression and mTOR silencing inhibited CML cell proliferation, promoted CML cell apoptosis, and sensitized these cells to imatinib. mTOR silencing reversed the promoting effect of miR-199a-3p inhibition on the proliferation of CML cells and the inhibitory effects on cell apoptosis and sensitivity to imatinib. MiR-199a-3p directly targeted mTOR. CONCLUSION: MiR-199a-3p suppressed cell propagation, facilitated apoptosis of CML cells, and sensitized CML cells to imatinib by downregulating mTOR signalling.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Apoptose , Bromodesoxiuridina/farmacologia , Bromodesoxiuridina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Luciferases/farmacologia , Luciferases/uso terapêutico , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Tirosina Quinases , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologiaRESUMO
In this study, the antitumor effects of tubulin-binding agent MPC-6827 on HeLa, MCF-7 and A549 cell lines originated from cervix carcinoma, metastatic breast adenocarcinoma and adenocarcinomic human alveolar basal epithelial cells respectively were determined. Cell index, BrdU labelling index, mitotic index and apoptotic index were evaluated in experiments. In cell index experiment 2 nM, 4 nM, 6 nM, 8 nM, 10 nM MPC-6827 applied to three cell lines. These parameters showed that 4 nM was the optimum concentration for HeLa and A549 cells, while 2 nM was the optimum concentration for MCF-7 cells. The use of optimum concentrations for each cell line has shown that while there was a significant decrease in mitotic index, BrdU labelling index, there was a significant increase in apoptotic index.
Assuntos
Antineoplásicos , Neoplasias , Quinazolinas , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Feminino , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacologiaRESUMO
OBJECTIVE: To investigate Sonic hedgehog (Shh) effects on proliferation and apoptosis of tongue epithelial cells in embryonic and ageing mice. MATERIALS AND METHODS: Embryonic day 13.5 (E13.5), E14.5, E16.5 and postnatal day 0.5 (PN0.5) K14-Cre;Shhfl/fl mice, and E14.5, E16.5, PN0.5, PN90.5 and postnatal 1.5 years (PN1.5Y) wild-type (Wt) mice were employed. Scanning electron microscopy, haematoxylin-eosin and immunohistochemistry were performed. Gel beads containing exogenous Shh protein were embedded in the tongue of PN90.5 and PN1.5Y Wt mice. Three days later, 5-bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA) immunohistochemical and TUNEL staining were performed. RESULTS: The number of fungiform papillae was decreased with age. The numbers of BrdU- and PCNA-positive cells were the highest at PN0.5 and the lowest at PN1.5Y. Compared with Wt mice, K14-Cre;Shhfl/fl mice had decreased PCNA-positive cells in the epithelium, a smaller tongue volume, and fewer papillae at PN0.5. At E14.5, the number of BrdU-positive cells was decreased in K14-Cre;Shh fl/fl mice. At PN1.5Y, the number of apoptotic cells in tongue tissue exposed to Shh protein was less than that in the BSA group and the numbers of BrdU- and PCNA-positive proliferating cells were increased. CONCLUSION: Shh maintains cell proliferation and reduces apoptosis during tongue development and ageing.
Assuntos
Proteínas Hedgehog , Transdução de Sinais , Animais , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Proliferação de Células , Células Epiteliais/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/farmacologia , Camundongos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/farmacologia , LínguaRESUMO
INTRODUCTION: Studies have suggested dexmedetomidine (DEX) as a potential antidepressant. However, no relevant research exists on its effects and mechanisms in curing depression caused by chronic pain. Therefore, an understanding of DEX's role in depressive disorders proposes new approaches for antidepressant treatment. METHODS: In this study, C57Bl/6 mice (n = 80) were divided into sham (n = 8) and chronic constrictive injury (CCI, n = 72) groups. The CCI group was further divided into six subgroups: CCI + normal saline (NS), CCI + DEX6.25, CCI + DEX12.5, CCI + DEX25, CCI + DEX50, and CCI + DEX100. Fourteen days after CCI, mice that did not develop a depressive phenotype were excluded through sucrose preference test (SPT), forced swimming test (FST), paw thermal withdrawal latency (PTWL), and serum corticosterone (CORT). Subsequently, mice in the sham group were administered 0.1 mL/10 g NS once daily. However, mice in the CCI subgroups were administered NS (0.1 mL/10 g), DEX (6.25 µg/kg), DEX (12.5 µg/kg), DEX (25 µg/kg), DEX (50 µg/kg), and DEX (100 µg/kg) intraperitoneally once daily for 1 week, respectively. Afterward, bromodeoxyuridine (BrdU) was injected intraperitoneally once daily as well for 3 consecutive days before sampling, following BrdU- and doublecortex (DCX)-positive cell detection in the hippocampus through immunofluorescence. RESULTS: The success rate of the chronic pain-depression (CPD) model was 62.5%. As observed, DEX dose-dependently affected sucrose preferences during the SPT and immobility time during FST. Results also showed that 25 µg/kg DEX had the best promotion effect during increased sucrose preference and reduced immobility time. Moreover, although DEX improved PTWL and serum CORT, no improvement over the DEX 25 µg/kg treatment was observed. Compared to the sham group, the percentage of BrdU+ and DCX+ cells was also significantly lower in the CCI + NS group. Besides, DEX dose-dependently affected cell proliferation and neuronal differentiation. Additionally, the percentage of BrdU+ and DCX+ cells in the dentate gyrus (DG) region of the hippocampus was highest in the CCI + DEX25 group. CONCLUSION: Therefore, DEX dose-dependently alleviates depression induced by chronic pain through neurogenesis promotion in the DG region of the hippocampus.
Assuntos
Dor Crônica , Dexmedetomidina , Neuralgia , Animais , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Bromodesoxiuridina/farmacologia , Corticosterona , Depressão/tratamento farmacológico , Dexmedetomidina/farmacologia , Dexmedetomidina/uso terapêutico , Hipocampo , Camundongos , Neuralgia/tratamento farmacológico , Neurogênese , Sacarose/farmacologiaRESUMO
Sevoflurane, a commonly used anesthetic, has been found to cause neural stem cell (NSC) injury, thereby contributing to neurocognitive impairment following general anesthesia. Tetramethylpyrazine (TMP), one of the most widely used medicinal compounds isolated from a traditional Chinese herb, possess neuroprotective activity. However, its effect on sevoflurane-induced NSC injury remains unclear. NSCs were pretreated with indicated concentrations of TMP for 2 h and then exposed to sevoflurane for 6 h. Cell injury was measured using lactate dehydrogenase (LDH) release assay. Cell viability and proliferation were detected by cell counting kit-8 (CCK-8) assay and 5-bromo-2'-deoxyuridine (BrdU) labeling, respectively. Apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The levels of cleaved caspase-3, phosphorylated protein kinase B (Akt) and phosphorylated glycogen synthase kinase-3ß (GSK-3ß) were detected by western blotting. Our results showed exposure to sevoflurane decreased the viability and proliferation of NSCs, while TMP preserved NSC viability and proliferation after sevoflurane exposure. In addition, the expression of cleaved caspase-3 and TUNEL positive cells were markedly decreased in TMP-treated NSCs compared with the control. Furthermore, pretreatment with TMP significantly increased the levels of phosphorylated Akt and GSK-3ß in sevoflurane-injured NSCs. However, an upstream inhibitor of Akt, LY294002 abolished the protective of TMP on the cell viability of NSCs. In conclusion, these findings indicate that TMP protects NSCs from sevoflurane-induced toxicity through Akt/GSK-3ß pathway.
Assuntos
Células-Tronco Neurais , Proteínas Proto-Oncogênicas c-akt , Ratos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sevoflurano/metabolismo , Sevoflurano/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Caspase 3/metabolismo , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidilexotransferase/farmacologia , Transdução de Sinais , Ratos Sprague-Dawley , Células-Tronco Neurais/metabolismo , Lactato Desidrogenases/metabolismo , ApoptoseRESUMO
An in vitro spermatogonial stem cell (SSC) culture can serve as an effective technique to study spermatogenesis and treatment for male infertility. In this research, we compared the effect of a three-dimensional alginate hydrogel with Sertoli cells in a 3D culture and co-cultured Sertoli cells. After harvest of SSCs from neonatal mice testes, the SSCs were divided into two groups: SSCs on a 3D alginate hydrogel with Sertoli cells and a co-culture of SSCs with Sertoli cells for 1 month. The samples were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and bromodeoxyuridine (BrdU) tracing, haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining after transplantation into an azoospermic testis mouse. The 3D group showed rapid cell proliferation and numerous colonies compared with the co-culture group. Molecular assessment showed significantly increased integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4 and Bcl2 expression levels in the 3D group and decreased expression levels of P53, Fas, and Bax. BrdU tracing, and H&E and PAS staining results indicated that the hydrogel alginate improved spermatogenesis after transplantation in vivo. This finding suggested that cultivation of SSCs on alginate hydrogel with Sertoli cells in a 3D culture can lead to efficient proliferation and maintenance of SSC stemness and enhance the efficiency of SSC transplantation.
Assuntos
Azoospermia , Células de Sertoli , Alginatos/metabolismo , Alginatos/farmacologia , Animais , Azoospermia/terapia , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Técnicas de Cocultura , Humanos , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Masculino , Camundongos , Espermatogônias , Células-Tronco , TestículoRESUMO
Neural progenitor cells (NPCs) transplantation is known as a potential strategy for treating spinal cord injury (SCI). This study aimed to investigate effects of insulin growth factor-1 (IGF-I) on NPCs proliferation and clarify associated mechanisms. NPCs isolated from T8-T10 segmental spinal cord tissues of rats were cultured and identification. Then, lentivirus packing plasmids containing IGF-I was constructed and used for NPCs infection. Cell proliferation was evaluated by detecting 5-Bromodeoxyuridine (BrdU) expression in NPCs, cell differentiation was detected using double-labeling immunofluorescence staining while cell apoptosis was detected using TUNEL assay. In addition, the signal expression of Akt/mTOR/p70S6K in NPCs cells were investigated using immunofluorescence staining and western blot assay. The experimental group was defined as pCMV-IGF-I group, while the negative control group was defined as pCMV-LacZ group. Cells infected with pCMV-IGF-I lentivirus followed by addition of 100 mg/ml rapamycin were defined as pCMV-IGF-I + Rapa group. NPCs were successfully isolated, identified and cultured. IGF-I overexpression significantly inhibited cell apoptosis and enhanced cell migration. Akt/mTOR/ p70S6K signaling cascade was proved to be present in NPCs, IGF-I overexpression significantly activated Akt/mTOR/p70S6K signaling cascade, while rapamycin addition inhibited its expression. Also, the activated Akt/mTOR/p70S6K signal cascade induced by IGF-I significantly enhanced BrdU expression and inhibited cell apoptosis, and promoted the differentiation of NPC into the neuronal system. However, the rapamycin addition inhibited the cell response induced by IGF-I overexpression. IGF-I overexpression could enhance cell proliferation, inhibit cell apoptosis and promote their differentiation into neuronal systems by activating Akt/mTOR/p70S6K signaling cascade in vitro, indicating that the Akt/mTOR/p70S6K signaling cascade may be the potentially mechanism for the endogenous repair and remodeling of spinal cord after injury.
Assuntos
Células-Tronco Neurais , Proteínas Quinases S6 Ribossômicas 70-kDa , Animais , Apoptose , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Proliferação de Células , Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Células-Tronco Neurais/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/farmacologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologiaRESUMO
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and warrants further study as well as timely treatment. Additionally, the mechanisms of the brain's intrinsic defense against chronic injury are not yet fully understood. Herein, we examined the response of the main neurogenic niches to amyloid exposure and the associated changes in structure and synaptic activity. Flow cytometry of Nestin-, Vimentin-, Nestin/Vimentin-, NeuN-, GFAP-, NeuN/GFAP-, NSE-, BrdU-, Wnt-, BrdU/Wnt-, VEGF-, Sox14-, VEGF/Sox14-, Sox10-, Sox2-, Sox10/Sox2-, Bax-, and Bcl-xL-positive cells was performed in the subventricular zone (SVZ), hippocampus, and cerebral cortex of rat brains on 90th day after intracerebroventricular (i.c.v.) single injection of a fraction of ß-amyloid (Aß) (1-42). The relative structural changes in these areas and disruptions to synaptic activity in the entorhinal cortex-hippocampus circuit were also evaluated. Our flow analyses revealed a reduction in the numbers of Nestin-, Vimentin-, and Nestin/Vimentin-positive cells in neurogenic niches and the olfactory bulb. These changes were accompanied by an increased number of BrdU-positive cells in the hippocampus and SVZ. The latter changes were strongly correlated with changes in the numbers of VEGF- and VEGF/Sox14-positive cells. The morphological changes were characterized by significant neural loss, a characteristic shift in entorhinal cortex-hippocampus circuit activity, and decreased spontaneous alternation in a behavioral test. We conclude that although an injection of Aß (1-42) induced stem cell proliferation and triggered neurogenesis at a certain stage, this process was incomplete and led to neural stem cell immaturity. We propose the idea of enhancing adult neurogenesis as a promising strategy for preventing dementia at healthy elderly people andpeople at high risk for developing AD, or treating patients diagnosed with AD.
Assuntos
Doença de Alzheimer , Fator A de Crescimento do Endotélio Vascular , Animais , Ratos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Neurogênese , Peptídeos beta-Amiloides/farmacologia , Encéfalo , Hipocampo , Bromodesoxiuridina/farmacologia , Proteínas Amiloidogênicas/farmacologiaRESUMO
Breast cancer is a highly heterogeneous disease that has been clinically divided into three main subtypes: estrogen receptor (ER)- and progesterone receptor (PR)-positive, human epidermal growth factor receptor 2 (HER 2)-positive, and triple-negative breast cancer (TNBC). With its high metastatic potential and resistance to endocrine therapy, HER 2-targeted therapy, and chemotherapy, TNBC represents an enormous clinical challenge. The genus Taraxacum is used to treat breast cancer in traditional medicine. Here, we applied aqueous extracts from two Taraxacum species, T. mongolicum and T. formosanum, to compare their potential antitumor effects against three human breast cancer cell lines: MDA-MB-231 (ER-, PR-, and HER2-), ZR-75-1 (ER+, PR+/-, and HER2-), and MCF-7 (ER+, PR+, and HER2-). Our results show that T. mongolicum exerted cytotoxic effects against MDA-MB-231 cells, including the induction of apoptosis, the reduction of cell proliferation, the disruption of the mitochondrial membrane potential, and/or the downregulation of the oxygen consumption rate. Both T. mongolicum and T. formosanum decreased cell migration and colony formation in the three cell-lines and exerted suppressive effects on MCF-7 cell proliferation based on metabolic activity and BrdU incorporation, but an enhanced proliferation of ZR-75-1 cells based on BrdU incorporation. T. formosanum induced ribotoxic stress in MDA-MB-231and ZR-75-1 cells; T. mongolicum did not. In summary, these findings suggest that T. mongolicum showed greater cytotoxicity against all three tested breast cancer cell lines, especially the TNBC MDA-MB-231 cell line.